Mechanisms for blood coagulation, thrombosis and haemostasis are well described in International Patent Publication WO 91/01382 the contents of which are incorporated herein by reference.
It is known from International Patent Publication WO 93/01261 and publications by Bertina et al 1994 and Dahlback et al 1995 that the risk of thrombosis in patients with a mutant factor V molecule known as the Leiden variant, or with activated protein C impairment for some other reason, may be determined by activating the coagulation system in a plasma sample and incubating the sample with activated protein C in what has come to be known as an activated protein C impairment, impedance or resistance test. There are precedents for this test in which impairment of activated protein C has been detected in patents with acquired thrombophilia (Mitchell et al, 1986; Amer et al. 1988).
New tests have recently been proposed to screen for most defects in the protein C pathway (PCP) thereby to rationalise the approach to individual assays for protein C, S and factor V(Leiden) which are currently requested together in all cases of thrombophilia investigation, with a very low rate of abnormality finding. These 2-stage clotting tests usually involve activating the patient's own plasma protein C either with thrombin/thrombomodulin complex or the activator from Agkistrodon Contortrix venom, commonly referred to as PROTAC™ of Pentapharm, Basle. This activated protein C (APC) then inactivates the patient's own factor Va in a protein S-dependent manner during a subsequent clotting test, yielding longer clotting times than if protein C had not been activated. Clotting times shorter than normal are obtained when defects in protein C and protein S occur as well as when APC resistant factor V(Leiden) is present. Such tests have been described based oil Activated Partial Thromboplastin Times (APTT) eg AU 28416/95, EP 718628 “Method for diagnosis of blood coagulation disorders”, dilute prothrombin time tests (PT) and WO 96/42018 “Thrombosis risk test”.
A substrate conversion reaction rate may be determined by the coagulation time or by the time required for the conversion of a chromogenic substrate to a coloured product. The conversion rate obtained is compared with values obtained in the absence of protein C activator or PCA and also with results on normal plasma samples. If the coagulation time is not sufficiently prolonged by protein C activator, it indicates that the individual from which the sample is derived may be at a higher-than-normal risk of thrombosis.
It is well known that activation of endogenous protein C in plasma by the activator from A. Contortrix venom prolongs subsequent clotting times to a degree related to the protein C content. Several other factors, however, influence or interfere with this test. These factors include protein S, factor V(Leiden) which are now recognised as thrombotic risk factors in their own right.
The present inventor has recently developed an improved APC resistance test which is described in WO 96/04560. This test requires the addition of exogenous reagents which activate factor V and activate the common pathway of the blood coagulation mechanism through factor X or by inducing the formation of thrombin in a factor V dependent manner together with exogenous APC to a plasma sample. It was found that if factor V is specifically activated by an exogenous reagent in addition to activation of the common pathway through factor X, the test for APC resistance may be made more sensitive and specific than previously known tests. The present inventor has also found that improved specificity is obtained when a complex factor X activator is used together with the factor V activator. This test, because the Russells viper venom contains activators of both factor X and factor V, has been referred to as the Russells Viper Venom (RVV)-based test. A similar result is achieved if prothrombin is activated to thrombin by a factor V dependent activator in the presence of a factor V activator such as those from Australian elapid venoms.
The protein C pathway is one of a number of antithrombotic mechanisms operating within normal blood vessels to control coagulation and prevent thrombosis. Probably the most important of these mechanisms is the glycosaminoglycan (GAG) pathway which requires antithrombin III as a cofactor and heparin cofactor 2. Thrombin and factor Xa are controlled by these two plasma inhibitors which are modulated by glycosaminoglycans such as heparin sulphates normally on endothelial cells lining healthy blood vessels.
The present inventor has made the surprising finding that such tests may be further modified to allow improved discrimination between healthy individuals and patients with impaired or aberrant blood anti-thrombotic mechanisms.